Gerhard, That's an interesting paper whose significance I had missed. We have not tested that property fo avidins. Oxidized guanosine has one ring that looks sort of like the urea part of biotin but I am surprised that the affinity is high enough because it has the ribose off one of those nitrogen atoms and the second aromatic ring of the purine bears little resemblence to the rest of biotin. We see nice selective staining of mitochondria by avidins in total absence of biotinylated probes so there could be difficulty staining 8-hydroxyguanosince in cells with avidins if there is only low-level damage. It is definitely a potential problem using any avidin or streptavidin conjugate with fixed cell preps that few people are aware of. [Photo: 46KB] The intermediate filaments in bovine pulmonary artery endothelial cells, localized using anti-desmin antibody (mouse monoclonal 131-15014), which was visualized with Alexa Fluor 647 goat anti–mouse IgG conjugate. Endogenous biotin in the mitochondria was labeled with an Alexa Fluor 546 streptavidin conjugate and DNA in the cell was stained with blue-fluorescent DAPI. Gerhard Nebe-von-Caron wrote: > It would be interesting to compare that with the paper of > http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9448838&dopt=Abstractas > they claim the "Direct detection of 8-oxodeoxyguanosine and 8-oxoguanine byavidin and > its analogues" as a measure of oxidative DNA damage. It looks verystraightforward > but suggests you should not block with streptavidin but justmeasure it's > binding.RegardsGerhard-----Original Message-----From: Richard Haugland > [SMTP:richard.haugland@probes.com]Sent: Friday, September 28, 2001 > 8:20 PMTo: Cytometry Mailing ListSubject: Re: Any thoughts on the detection > of Ionizing radiationeffects in ratsThere is a possibility that the reagent > ARPhttp://www.probes.com/servlets/product?region=Select+Region&item=10550would > be useful.This biotin derivative reacts with abasic sites of nucleic acids > (nucleic acidsthat havelost their base but not the backbone structure > and thus generate aldehydes.ARP has been used to detect this event in living > cells:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10639140&dopt=Abstractand > abasic sites produced by reactive-oxygen > specieshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1567824&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8347625&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11020331&dopt=AbstractIonizing > radiation also produces abasic sites. Use of a green-fluorescentstreptavidinconjugate > should work in flow cytometry (or imaging); however, I recommend thatyou blockendogenous > biotin first with streptavidin followed by excess biotin.Mitochondria, inparticular, > have endogenous biotinylated proteins that could interfere withlow-leveldetection. Cells > with endogenous damage by ROS may also react as positive so youwill needcontrols.Simon > Monard wrote:> Hi> Could you look for chomosome damage by flow cytometry. I don't > know how rarechromosome> damage would be. Its not very hard to prepare chomsome preps > from rats,either con> A stimulated blood or perhaps you could look at granukoma pouch > assay cells.Just> a thought. We made some chromosome paints for rat and tried to look > forabnormal> chomosomes by microscopy also.> Simon> FLOWers>> I am in need of > some help.>> We want to look at the protective effect of drug X on radiation induced> > damage in the rat, by Flow Cytometry.> Does anyone have any thoughts??>> I know this > is potentially a very broad subject, but I would appreciate any> thoughts on potential > avenues of approach> > > thank you for your time and help > > > > Philip Barren
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